Enterocin A结构基因和免疫基因的克隆及序列特征分析

Cloning and Nucleotide Sequence of Genes Involved in Enterocin A Production and Immunity

采用PCR技术从屎肠球菌的染色体DNA上扩增到1条529bp的DNA片段entAIc,以pGEM-T为载体,将该基因片段克隆到大肠杆菌中,对阳性重组质粒pGAI测序。序列分析结果表明片段entAIc含有2个ORF,EnterocinA结构基因entA和免疫基因entI,2个基因紧密相连。核苷酸序列分析显示,不同菌株来源的entA和entI具有较高的同源性,分别达到99.87%和99.76%。氨基酸序列分析显示不同菌株来源的EnterocinA前体完全相同,免疫蛋白仅有1个氨基酸差异。

Chromosomal DNA was extracted from Enterococcus faecium AS 1.2025.A 529 bp fragment entAIc was obtained using PCR.The PCR product was cloned into pGEM-T,resulting in plasmid pGAI,DNA sequencing analysis of pGAI revealed that the presence of two consecutive open reading frames(ORFs).The first ORF entA,encoding the enterocin A prepeptide of 65 amino acid residues,consisted of 198 nucleotides;the second ORF entI,spanning 312 bp,was located 2 nucleotides downstream of entA and encoded a 103-amino-acid protein conferring immunity to enterocin A.Sequence alignment showed that entA and entI from different E.faecium strains shared 99.87% and 99.76% identity,respectively;Enterocin A prepeptide exhibited complete identity,while immunity protein diversity of 1 amino acid residue.